B cells are an integral component in mounting humoral immune responses and they are also crucial in programming T cell mediated immunity. Usually, B cell activation is initiated by recognition of antigen through B cell receptor (BCR), followed by its processing and presentation to T cells. But very little is known about BCR independent activation of B cells. Now, there is an increasing body of evidence indicating the combinatorial effect of innate and adaptive immune components in modulating the functions of B cells. In this study, we demonstrate the activation of resting B cells (RB) by simultaneous involvement of Toll like Receptor-2 (TLR-2) and costimulatory molecule, CD86. Interestingly, these B cells exhibited significant level of activation and proliferation. Furthermore, this process of activation leads to the differentiation of RB cells, preferably into marginal zone precursors (CD19+IgDhiIgMhiCD21/35hiCD23hi) in a shorter time window and showed increased secretion of IgG isotype. These RB cells also showed enhanced antigen uptake capacity. These observations were also substantiated by microarray gene expression results, which strengthen the notion that combinatorial signaling through innate and adaptive immune components enhances B cell mediated immune response. Thus, the present study elucidates a novel BCR independent B cell activation mechanism that links TLR-2 and CD86. Hence signaling of TLRs in conjunction with costimulatory molecules will substantially help in bolstering humoral immune response, which can be extrapolated to formulate vaccination strategies for diseases involving B cell-mediated immunity.
Some of the most successful pathogens of human, such as Mycobacterium tuberculosis (Mtb), HIV, and Leishmania donovani not only establish chronic infections but also remain a grave global threat. These pathogens have developed innovative strategies to evade immune responses such as antigenic shift and drift, interference with antigen processing/presentation, subversion of phagocytosis, induction of immune regulatory pathways, and manipulation of the costimulatory molecules. Costimulatory molecules expressed on the surface of various cells play a decisive role in the initiation and sustenance of immunity. Exploitation of the “code of conduct” of costimulation pathways provides evolutionary incentive to the pathogens and thereby abates the functioning of the immune system. Here we review how Mtb, HIV, Leishmania sp., and other pathogens manipulate costimulatory molecules to establish chronic infection. Impairment by pathogens in the signaling events delivered by costimulatory molecules may be responsible for defective T-cell responses; consequently organisms grow unhindered in the host cells. This review summarizes the convergent devices that pathogens employ to tune and tame the immune system using costimulatory molecules. Studying host-pathogen interaction in context with costimulatory signals may unveil the molecular mechanism that will help in understanding the survival/death of the pathogens. We emphasize that the very same pathways can potentially be exploited to develop immunotherapeutic strategies to eliminate intracellular pathogens.
Molecular mimicry between microbial antigens and host-proteins is one of the etiological enigmas for the occurrence of autoimmune diseases. T cells that recognize cross-reactive epitopes may trigger autoimmune reactions. Intriguingly, autoimmune diseases have been reported to be prevalent in tuberculosis endemic populations. Further, association of Mycobacterium tuberculosis (M. tuberculosis) has been implicated in different autoimmune diseases, including rheumatoid arthritis and multiple sclerosis. Although, in silico analyses have identified a number of M. tuberculosis specific vaccine candidates, the analysis on prospective cross-reactive epitopes, that may elicit autoimmune response, has not been yet attempted. Here, we have employed bioinformatics tools to determine T cell epitopes of homologous antigenic regions between M. tuberculosis and human proteomes.
Employing bioinformatics tools, we have identified potentially cross-reactive T cell epitopes restricted to predominant class I and II alleles of human leukocyte antigens (HLA). These are similar to peptides of mycobacterial proteins and considerable numbers of them are promiscuous. Some of the identified antigens corroborated with established autoimmune diseases linked with mycobacterial infection.
The present study reveals many target proteins and their putative T cell epitopes that might have significant application in understanding the molecular basis of possible T cell autoimmune reactions during M. tuberculosis infections.
Conventionally, signaling through BCR initiates sequence of events necessary for activation and differentiation of B cells. We report an alternative approach, independent of BCR, for stimulating resting B (RB) cells, by involving TLR-2 and CD40 - molecules crucial for innate and adaptive immunity. CD40 triggering of TLR-2 stimulated RB cells significantly augments their activation, proliferation and differentiation. It also substantially ameliorates the calcium flux, antigen uptake capacity and ability of B cells to activate T cells. The survival of RB cells was improved and it increases the number of cells expressing activation induced deaminase (AID), signifying class switch recombination (CSR). Further, we also observed increased activation rate and decreased threshold period required for optimum stimulation of RB cells. These results corroborate well with microarray gene expression data. This study provides novel insights into coordination between the molecules of innate and adaptive immunity in activating B cells, in a BCR independent manner. This strategy can be exploited to design vaccines to bolster B cell activation and antigen presenting efficiency, leading to faster and better immune response.
BCG has been administered globally for more than 75 years, yet tuberculosis (TB) continues to kill more than 2 million people annually. Further, BCG protects childhood TB but is quite inefficient in adults. This indicates that BCG fails to induce long-term protection. Hence there is a need to explore alternative vaccination strategies that can stimulate enduring T cell memory response. Dendritic cell based vaccination has attained extensive popularity following their success in various malignancies. In our previous study, we have established a novel and unique vaccination strategy against Mycobacterium tuberculosis (M. tb) and Salmonella typhimurium by utilizing infected macrophages (IM). In short-term experiments (30 days), substantial degree of protection was observed. However, remarkable difference was not observed in long-term studies (240 days) due to failure of the vaccine to generate long-lasting memory T cells. Hence, in the present study we employed T cell memory augmenting cytokines IL-1+IL-6+TNF-α and IL-7+IL-15 for the induction of the enhancement of long-term protection by the vaccine. We co-administered the M. tb infected macrophages vaccine with IL-1+IL-6+TNF-α (IM-1.6.α) and IL-7+IL-15 (IM-7.15). The mice were then rested for a reasonably large period (240 days) to study the bona fide T cell memory response before exposing them to aerosolized M. tb. IM-1.6.α but not IM-7.15 significantly improved memory T cell response against M. tb, as evidenced by recall responses of memory T cells, expansion of both central as well as effector memory CD4 and CD8 T cell pools, elicitation of mainly Th1 memory response, reduction in the mycobacterial load and alleviated lung pathology. Importantly, the protection induced by IM-1.6.α was significantly better than BCG. Thus, this study demonstrates that not only antigen-pulsed DCs can be successfully employed as vaccines against cancer and infectious diseases but also macrophages infected with M. tb can be utilized with great efficacy especially in protection against TB.
We have outlined the carefully orchestrated process of CD4+ T-cell differentiation from naïve to effector and from effector to memory cells with a focus on how these processes can be studied in vivo in responses to pathogen infection. We emphasize that the regulatory factors that determine the quality and quantity of the effector and memory cells generated include (i) the antigen dose during the initial T-cell interaction with antigen-presenting cells; (ii) the dose and duration of repeated interactions; and (iii) the milieu of inflammatory and growth cytokines that responding CD4+ T cells encounter. We suggest that heterogeneity in these regulatory factors leads to the generation of a spectrum of effectors with different functional attributes. Furthermore, we suggest that it is the presence of effectors at different stages along a pathway of progressive linear differentiation that leads to a related spectrum of memory cells. Our studies particularly highlight the multi-faceted roles of CD4+ effector and memory T cells in protective responses to influenza infection and support the concept that efficient priming of CD4+ T cells that react to shared influenza proteins could contribute greatly to vaccine strategies for influenza.
Presence of antigen presenting cells, expression of costimulatory molecules, the strength of first signal and cytokine milieu are quite important in influencing the reactivation of differentiated Th1 and Th2 cells.
In the present study, we have analyzed the concerted action of pro-Th1 and pro-Th2 cytokines in the presence of B cells, peritoneal and splenic macrophages as antigen presenting cells and varied concentration of first (anti-CD3 Ab) and second (B7-1 transfectant) signals on the proliferation and cytokine secretion by Th1 and Th2 cells. Interesting observations were made that IFN-γ significantly augmented the secretion of IL-4 by Th2 cells when either B cells or splenic or peritoneal macrophages were used as APC. Further, IFN-γ significantly inhibited the proliferation of Th1 cells only in the presence of peritoneal macrophages. We have also observed that B cells could significantly respond to cytokines to further enhance the proliferation and cytokine release by Th1 and Th2 cells. But not much effect on addition of exogenous cytokines IL-1, IL-4, IL-5, IL-12 was observed on the proliferation of Th1 and Th2 cells in the presence of macrophages. In contrast, both IFN-γ and IL-2 significantly enhanced the production of IL-4 and IL-5 respectively, by Th2 cells in presence of B cells, splenic and peritoneal macrophages. Another important observation was that the addition of B7-1 transfectants in the cultures, which were stimulated with low dose of anti-CD3 Ab significantly, enhanced the proliferation and cytokine secretion.
This study indicates involvement of different type of APCs, cytokine milieu, dose of first and second signals in a concerted manner in the outcome of the immune response. The significance of this study is that the immunization with antigen along with costimulatory molecules may significantly reduce the dose of antigen and can generate better immune response than antigen alone.
Cluster of differentiation 14 (CD14) gene is an important component of the human innate immune system and its role in tuberculosis (TB) has been sparsely documented. The enhanced plasma CD14 levels in TB patients as compared to healthy controls are associated with CD14 gene promoter (C-159T) polymorphism. In the past few years, the relationship between CD14 −159 C>T (rs2569190) polymorphism and risk of TB has been reported in various ethnic populations; however, those studies have yielded contradictory results. In this study systemic assessment was done for the published studies based on the association between CD14 −159 C>T polymorphism and TB risk retrieved from PubMed (Medline) and EMBASE search. A total number of 1389 TB cases and 1421 controls were included in this study and meta-analysis was performed to elucidate the association between CD14 −159 C>T polymorphism and its susceptibility towards TB. Pooled odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated for allele contrast, homozygous comparison, heterozygous comparison, dominant and recessive genetic model. It was found that T allele carrier was significantly associated with increased TB risk (T vs. C: p-value = 0.023; OR = 1.305, 95% CI = 1.038 to 1.640). Similarly, homozygous mutant TT genotype also revealed 1.6 fold increased risk of TB (TT vs. CC; p-value = 0.040; OR = 1.652, 95% CI = 1.023 to 2.667). Additionally, dominant genetic model demonstrated increased risk of developing TB (TT vs. CC+CT: p-value = 0.006; OR = 1.585, 95% CI = 1.142 to 2.201). The study demonstrates that CD14 gene (−159 C>T) polymorphism contributes increased susceptibility for TB. Moreover, this meta-analysis also suggests for future larger studies with stratified case control population and biological characterization for validation studies.
Ureaplasma species are the most frequently isolated microorganisms inside the amniotic cavity and have been associated with spontaneous abortion, chorioamnionitis, premature rupture of the membranes (PROM), preterm labour (PL) pneumonia in neonates and bronchopulmonary dysplasia in neonates. The mechanisms by which Ureaplasmas cause such diseases remain unclear, but it is believed that inappropriate induction of inflammatory responses is involved, triggered by the innate immune system. As part of its mechanism of activation, the innate immune system employs germ-lined encoded receptors, called pattern recognition receptors (PRRs) in order to “sense” pathogens. One such family of PRRs are the Toll like receptor family (TLR). In the current study we aimed to elucidate the role of TLRs in Ureaplasma-induced inflammation in human amniotic epithelial cells. Using silencing, as well as human embryonic kidney (HEK) transfected cell lines, we demonstrate that TLR2, TLR6 and TLR9 are involved in the inflammatory responses against Ureaplasma parvum and urealyticum serovars. Ureaplasma lipoproteins, such as Multiple Banded antigen (MBA), trigger responses via TLR2/TLR6, whereas the whole bacterium is required for TLR9 activation. No major differences were observed between the different serovars. Cell activation by Ureaplasma parvum and urealyticum seem to require lipid raft function and formation of heterotypic receptor complexes comprising of TLR2 and TLR6 on the cell surface and TLR9 intracellularly.
Macrophage C-type lectin (MCL) is a membrane surface receptor encoded by the Antigen Presenting Lectin-like gene Complex (APLEC). We generated a mouse monoclonal antibody for the study of this receptor in the rat. We demonstrate that rat MCL is expressed on blood monocytes and neutrophils, as well as on several tissue macrophage populations, including alveolar and peritoneal cavity macrophages. We also demonstrate MCL expression on a subset of resident spleen macrophages. Immunohistochemistry analysis of the spleen showed staining specifically in the marginal zone and red pulp. Exposure to pro-inflammatory mediators or to yeast cell wall extract (zymosan) increased surface MCL expression on peritoneal macrophages. We characterized a rat myeloid cell line, RMW, which expresses high levels of MCL. We found that MCL co-immunoprecipitated with the activating adaptor protein FcεRIγ in these cells. Moreover, beads coated with anti-MCL antibody increased phagocytosis in the RMW cells. Together, these observations indicate that rat MCL is a receptor that activates phagocytosis in myeloid cells under inflammatory conditions.
Functional T-cell responses are initiated by physical interactions between T-cells and antigen-presenting cells (APCs), including dendritic cells (DCs) and B-cells. T-cells are activated more effectively by DCs than by B-cells, but little is known about the key molecular mechanisms that underpin the particular potency of DC in triggering T-cell responses. To better understand the influence of physical intercellular interactions on APC efficacy in activating T-cells, we used single cell force spectroscopy to characterize and compare the mechanical forces of interactions between DC:T-cells and B:T-cells. Following antigen stimulation, intercellular interactions of DC:T-cell conjugates were stronger than B:T-cell interactions. DCs induced higher levels of T-cell calcium mobilization and production of IL-2 and IFNγ than were elicited by B-cells, thus suggesting that tight intercellular contacts are important in providing mechanically stable environment to initiate T-cell activation. Blocking antibodies targeting surface co-stimulatory molecules CD80 or CD86 weakened intercellular interactions and dampen T-cell activation, highlighting the amplificatory roles of CD80/86 in regulating APC:T-cell interactions and T-cell functional activation. The variable strength of mechanical forces between DC:T-cells and B:T-cell interactions were not solely dependent on differential APC expression of CD80/86, since DCs were superior to B-cells in promoting strong interactions with T-cells even when CD80 and CD86 were inhibited. These data provide mechanical insights into the effects of co-stimulatory molecules in regulating APC:T-cell interactions.
Mycobacterium tuberculosis is a major human pathogen that has evolved survival mechanisms to persist in an immune-competent host under a dormant condition. The regulation of M. tuberculosis metabolism during latent infection is not clearly known. The dormancy survival regulon (DosR regulon) is chiefly responsible for encoding dormancy related functions of M. tuberculosis. We describe functional characterization of an important gene of DosR regulon, Rv0079, which appears to be involved in the regulation of translation through the interaction of its product with bacterial ribosomal subunits. The protein encoded by Rv0079, possibly, has an inhibitory role with respect to protein synthesis, as revealed by our experiments. We performed computational modelling and docking simulation studies involving the protein encoded by Rv0079 followed by in vitro translation and growth curve analysis experiments, involving recombinant E. coli and Bacille Calmette Guérin (BCG) strains that overexpressed Rv0079. Our observations concerning the interaction of the protein with the ribosomes are supportive of its role in regulation/inhibition of translation. We propose that the protein encoded by locus Rv0079 is a ‘dormancy associated translation inhibitor’ or DATIN.
The role of bone marrow-derived mesenchymal stem cells (MSC) in the physiology of the gastrointestinal tract epithelium is currently not well established. These cells can be recruited in response to inflammation due to epithelial damage, home, and participate in tissue repair. In addition, in the case of tissue repair failure, these cells could transform and be at the origin of carcinomas. However, the chemoattractant molecules responsible for MSC recruitment and migration in response to epithelial damage, and particularly to Helicobacter pylori infection, remain unknown although the role of some chemokines has been suggested. This work aimed to get insight into the mechanisms of mouse MSC migration during in vitro infection of mouse gastrointestinal epithelial cells by H. pylori. Using a cell culture insert system, we showed that infection of gastrointestinal epithelial cells by different H. pylori strains is able to stimulate the migration of MSC. This mechanism involves the secretion by infected epithelial cells of multiple cytokines, with a major role of TNFα, mainly via a Nuclear Factor-kappa B-dependent pathway. This study provides the first evidence of the role of H. pylori infection in MSC migration and paves the way to a better understanding of the role of bone marrow-derived stem cells in gastric pathophysiology and carcinogenesis.
Neisseria meningitidis is a human pathogen responsible for life-threatening inflammatory diseases. Meningococcal penicillin-binding proteins (PBPs) and particularly PBP2 are involved in bacterial resistance to β-lactams. Here we describe a novel function for PBP2 that activates human and mouse dendritic cells (DC) in a time and dose-dependent manner. PBP2 induces MHC II (LOGEC50 = 4.7 µg/ml±0.1), CD80 (LOGEC50 = 4.88 µg/ml±0.15) and CD86 (LOGEC50 = 5.36 µg/ml±0.1). This effect was abolished when DCs were co-treated with anti-PBP2 antibodies. PBP2-treated DCs displayed enhanced immunogenic properties in vitro and in vivo. Furthermore, proteins co-purified with PBP2 showed no effect on DC maturation. We show through different in vivo and in vitro approaches that this effect is not due to endotoxin contamination. At the mechanistic level, PBP2 induces nuclear localization of p65 NF-kB of 70.7±5.1% cells versus 12±2.6% in untreated DCs and needs TLR4 expression to mature DCs. Immunoprecipitation and blocking experiments showed that
PBP2 binds TLR4. In conclusion, we describe a novel function of meningococcal PBP2 as a pathogen associated molecular pattern (PAMP) at the host-pathogen interface that could be recognized by the immune system as a danger signal, promoting the development of immune responses.
Burkholderia cepacia is a Gram-negative pathogen that causes serious respiratory infections in immunocompromised patients and individuals with cystic fibrosis. This bacterium is known to release extracellular proteins that may be involved in virulence.
In the present study, B. cepacia grown to mid-logarithmic and early-stationary phases were investigated on their ability to invade and survive intracellularly in A549 lung epithelial cells in order to discern the fate of these bacteria in the pathogenesis of B. cepacia lung infections in in vitro condition. The early-stationary phase B. cepacia was demonstrated to be more invasive than mid-logarithmic phase. In addition, culture supernatants of B. cepacia obtained from these phases of growth were also demonstrated to cause different cytotoxic potency on the A549 human lung epithelial cells. Profiling of the supernatants using the gel-based proteomics approach identified 43 proteins that were commonly released in both the growth phases and 40 proteins newly-released at the early-stationary phase. The latter proteins may account for the higher cytotoxic activity of the early-stationary culture supernatant compared to that obtained at the mid-logarithmic phase. Among the newly-released proteins in the early-stationary phase supernatant were flagellar hook-associated domain protein (FliD), flagellar hook-associated protein (FlgK), TonB-dependent siderophore (Fiu), Elongation factor G (FusA), phosphoglycerate kinase (Pgk) and sulfatase (AslA) which are known for their virulence.
Differences in the ability of B. cepacia to invade and survive intracellularly inside the epithelial cells at different phases of growth may improve our understanding of the varied disease progressions associated with B. cepacia infections. In addition, the identified culture supernatant proteins may be used as targets for the development of new strategies to control B. cepacia infection using agents that can block their release.
A comparative genomics approach was utilised to compare the genomes of Mycobacterium avium subspecies paratuberculosis (MAP) isolated from early onset paediatric Crohn's disease (CD) patients as well as Johne's diseased animals. Draft genome sequences were produced for MAP isolates derived from four CD patients, one ulcerative colitis (UC) patient, and two non-inflammatory bowel disease (IBD) control individuals using Illumina sequencing, complemented by comparative genome hybridisation (CGH). MAP isolates derived from two bovine and one ovine host were also subjected to whole genome sequencing and CGH. All seven human derived MAP isolates were highly genetically similar and clustered together with one bovine type isolate following phylogenetic analysis. Three other sequenced isolates (including the reference bovine derived isolate K10) were genetically distinct. The human isolates contained two large tandem duplications, the organisations of which were confirmed by PCR. Designated vGI-17 and vGI-18 these duplications spanned 63 and 109 open reading frames, respectively. PCR screening of over 30 additional MAP isolates (3 human derived, 27 animal derived and one environmental isolate) confirmed that vGI-17 and vGI-18 are common across many isolates. Quantitative real-time PCR of vGI-17 demonstrated that the proportion of cells containing the vGI-17 duplication varied between 0.01 to 15% amongst isolates with human isolates containing a higher proportion of vGI-17 compared to most animal isolates. These findings suggest these duplications are transient genomic rearrangements. We hypothesise that the over-representation of vGI-17 in human derived MAP strains may enhance their ability to infect or persist within a human host by increasing genome redundancy and conferring crude regulation of protein expression across biologically important regions.
Helicobacter pylori induces cytokine mediated changes in gastroduodenal pathophysiology, wherein, the activated macrophages at the sub-mucosal space play a central role in mounting innate immune response against the antigens. The bacterium gains niche through persistent inflammation and local immune-suppression causing peptic ulcer disease or chronic gastritis; the latter being a significant risk factor for the development of gastric adenocarcinoma. What favors persistence of H. pylori in the gastric niches is not clearly understood. We report detailed characterization of a functionally unknown gene (HP986), which was detected in patient isolates associated with peptic ulcer and gastric carcinoma. Expression and purification of recombinant HP986 (rHP986) revealed a novel, ∼29 kDa protein in biologically active form which associates with significant levels of humoral immune responses in diseased individuals (p<0.001). Also, it induced significant levels of TNF-α and Interleukin-8 in cultured human macrophages concurrent to the translocation of nuclear transcription factor-κB (NF-κB). Further, the rHP986 induced apoptosis of cultured macrophages through a Fas mediated pathway. Dissection of the underlying signaling mechanism revealed that rHP986 induces both TNFR1 and Fas expression to lead to apoptosis. We further demonstrated interaction of HP986 with TNFR1 through computational and experimental approaches. Independent proinflammatory and apoptotic responses triggered by rHP986 as shown in this study point to its role, possibly as a survival strategy to gain niche through inflammation and to counter the activated macrophages to avoid clearance.
Current chemotherapeutic drugs kill cancer cells mainly by inducing apoptosis. However, they become ineffective once cancer cell has the ability to metastasize, hence the poor prognosis and high mortality rate. Therefore, the purpose of this study was to evaluate the antimetastatic potential of Phyllanthus (P. niruri, P. urinaria, P. watsonii, and P. amarus) on lung and breast carcinoma cells.
Cytotoxicity of Phyllanthus plant extracts were first screened using the MTS reduction assay. They were shown to inhibit MCF-7 (breast carcinoma) and A549 (lung carcinoma) cells growth with IC50 values ranging from 50–180 µg/ml and 65–470 µg/ml for methanolic and aqueous extracts respectively. In comparison, they have lower toxicity on normal cells with the cell viability percentage remaining above 50% when treated up to 1000 µg/ml for both extracts. After determining the non-toxic effective dose, several antimetastasis assays were carried out and Phyllanthus extracts were shown to effectively reduce invasion, migration, and adhesion of both MCF-7 and A549 cells in a dose-dependent manner, at concentrations ranging from 20–200 µg/ml for methanolic extracts and 50–500 µg/ml for aqueous extracts. This was followed by an evaluation of the possible modes of cell death that occurred along with the antimetastatic activity. Phyllanthus was shown to be capable of inducing apoptosis in conjunction with its antimetastastic action, with more than three fold increase of caspases-3 and -7, the presence of DNA-fragmentation and TUNEL-positive cells. The ability of Phyllanthus to exert antimetastatic activities is mostly associated to the presence of polyphenol compounds in its extracts.
The presence of polyphenol compounds in the Phyllanthus plant is critically important in the inhibition of the invasion, migration, and adhesion of cancer cells, along with the involvement of apoptosis induction. Hence, Phyllanthus could be a valuable candidate in the treatment of metastatic cancers.
B cells play an important role in the pathogenesis of both systemic and organ-specific autoimmune diseases. Autoreactive B cells not only produce autoantibodies, but also are capable to efficiently present specific autoantigens to T cells. Furthermore, B cells can secrete proinflammatory cytokines and amplify the vicious process of self-destruction. B cell-directed therapy is a potentially important approach for treatment of various autoimmune diseases. The depletion of B cells by anti-CD20/19 monoclonal antibody Retuximab® used in autoimmune diseases therapy leads to systemic side effects and should be significantly improved. In this study we designed a repertoire of genetically engineered B cell killers that specifically affected one kind of cells carrying a respective B cell receptor. We constructed immunotoxins (ITs), fused with c-myc epitope as a model targeting sequence, based on barnase, Pseudomonas toxin, Shiga-like toxin E.coli and Fc domain of human antibody IgGγ1. C-MYC hybridoma cell line producing anti-c-myc IgG was chosen as a model for targeted cell depletion. C-myc sequence fused with toxins provided addressed delivery of the toxic agent to the target cells. We demonstrated functional activity of designed ITs in vitro and showed recognition of the fusion molecules by antibodies produced by targeted hybridoma. To study specificity of the proposed B cells killing molecules, we tested a set of created ITs ex vivo, using C-MYC and irrelevant hybridoma cell lines. Pseudomonas-containing IT showed one of the highest cytotoxic effects on the model cells, however, possessed promiscuous specificity. Shiga-like toxin construct demonstrated mild both cytotoxicity and specificity. Barnase and Fc-containing ITs revealed excellent balance between their legibility and toxic properties. Moreover, barnase and Fc molecules fused with c-myc epitope were able to selectively deplete c-myc-specific B cells and decrease production of anti-c-myc antibodies in culture of native splenocytes, suggesting their highest therapeutic potential as targeted B cell killing agents.
Many efforts have been made to understand basal mechanisms of mycobacterial infections. Macrophages are the first line of host immune defence to encounter and eradicate mycobacteria. Pathogenic species have evolved different mechanisms to evade host response, e.g. by influencing macrophage apoptotic pathways. However, the underlying molecular regulation is not fully understood. A new layer of eukaryotic regulation of gene expression is constituted by microRNAs. Therefore, we present a comprehensive study for identification of these key regulators and their targets in the context of host macrophage response to mycobacterial infections.
We performed microRNA as well as mRNA expression analysis of human monocyte derived macrophages infected with several Mycobacterium avium hominissuis strains by means of microarrays as well as quantitative reverse transcription PCR (qRT-PCR). The data revealed the ability of all strains to inhibit apoptosis by transcriptional regulation of BCL2 family members. Accordingly, at 48 h after infection macrophages infected with all M. avium strains showed significantly decreased caspase 3 and 7 activities compared to the controls. Expression of let-7e, miR-29a and miR-886-5p were increased in response to mycobacterial infection at 48 h. The integrated analysis of microRNA and mRNA expression as well as target prediction pointed out regulative networks identifying caspase 3 and 7 as potential targets of let-7e and miR-29a, respectively. Consecutive reporter assays verified the regulation of caspase 3 and 7 by these microRNAs.
We show for the first time that mycobacterial infection of human macrophages causes a specific microRNA response. We furthermore outlined a regulatory network of potential interactions between microRNAs and mRNAs. This study provides a theoretical concept for unveiling how distinct mycobacteria could manipulate host cell response. In addition, functional relevance was confirmed by uncovering the control of major caspases 3 and 7 by let-7e and miR-29a, respectively.
T cells are major players of adaptive immune response in mammals. Recognition of
an antigenic peptide in association with the major histocompatibility complex at
the surface of an antigen presenting cell (APC) is a specific and sensitive
process whose mechanism is not fully understood. The potential contribution of
mechanical forces in the T cell activation process is increasingly debated,
although these forces are scarcely defined and hold only limited experimental
evidence. In this work, we have implemented a biomembrane force probe (BFP)
setup and a model APC to explore the nature and the characteristics of the
mechanical forces potentially generated upon engagement of the T cell receptor
(TCR) and/or lymphocyte function-associated antigen-1 (LFA-1). We show that upon
contact with a model APC coated with antibodies towards TCR-CD3, after a short
latency, the T cell developed a timed sequence of pushing and pulling forces
against its target. These processes were defined by their initial constant
growth velocity and loading rate (force increase per unit of time). LFA-1
engagement together with TCR-CD3 reduced the growing speed during the pushing
phase without triggering the same mechanical behavior when engaged alone.
Intracellular Ca2+ concentration
([Ca2+]i) was monitored simultaneously
to verify the cell commitment in the activation process.
[Ca2+]i increased a few tens of seconds
after the beginning of the pushing phase although no strong correlation appeared
between the two events. The pushing phase was driven by actin polymerization.
Tuning the BFP mechanical properties, we could show that the loading rate during
the pulling phase increased with the target stiffness. This indicated that a
mechanosensing mechanism is implemented in the early steps of the activation
process. We provide here the first quantified description of force generation
sequence upon local bidimensional engagement of TCR-CD3 and discuss its
potential role in a T cell mechanically-regulated activation process.
Ferritins are recognized as key players in the iron storage and detoxification processes. Iron acquisition in the case of pathogenic bacteria has long been established as an important virulence mechanism. Here, we report a 3.0 Å crystal structure of a ferritin, annotated as Bacterioferritin B (BfrB), from Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis that continues to be one of the world's deadliest diseases. Similar to the other members of ferritin family, the Mtb BfrB subunit exhibits the characteristic fold of a four-helical bundle that possesses the ferroxidase catalytic centre. We compare the structure of Mtb BfrB with representatives of the ferritin family belonging to the archaea, eubacteria and eukarya. Unlike most other ferritins, Mtb BfrB has an extended C-terminus. To dissect the role of this extended C-terminus, truncated Mtb BfrB was purified and biochemical studies implicate this region in ferroxidase activity and iron release in addition to providing stability to the protein. Functionally important regions in a protein of known 3D-structure can be determined by estimating the degree of conservation of the amino-acid sites with its close homologues. Based on the comparative studies, we identify the slowly evolving conserved sites as well as the rapidly evolving variable sites and analyze their role in relation to structure and function of Mtb BfrB. Further, electrostatic computations demonstrate that although the electrostatic environment of catalytic residues is preserved within the family, extensive variability is exhibited by residues defining the channels and pores, in all likelihood keeping up with the diverse functions executed by these ferritins in varied environments.